In the technology of separation of compounds using chromatography, analyzing and collecting separated fractions of chemical compounds have advanced to processes and systems that vary greatly in process, instruments, and production capability. Liquid chromatography uses a fluid, called a mobile phase, that is pumped through a chromatography separation column, called the stationary phase. A liquid sample containing one or more chemical compounds is injected into the mobile phase flowstream at the head of the column. Different compounds in the sample are delayed in the stationary phase for different time periods.
The separated compounds exit the column according to different retention times. These separated compounds can be detected and graphed as a “peak” of the injected sample. Purified samples can then be collected that correspond to the peak in the flowstream. If constant temperature, pressure, flowrate, and injection sample composition is maintained in the system, then repeated injections of the compounds to separate into the column can produce repeatable peaks eluting from the column. These eluted peaks contain purified analytes of interest that can be collected through automated timing of a collection system.
The parameters and instruments of the chromatography system can be adjusted in order to optimize the speed, efficiency, and accuracy of analyte collection. An advancement of LC is HPLC (High Performance Liquid Chromatography), which uses 20 mm to one inch diameter columns with flowrates optimized at 20 to 30 ml/min. While process and collection speeds are faster than LC, drawbacks to HPLC include high waste solvent production and slow effective process time for samples due to removal of solvent and water from collected sample fractions.
In FIG. 1, a pump system 10 feeds a mobile phase under pressure into packed chromatography column 14. Injection valve 12 injects sample at the head of column 14. The efficiency of the separation of sample components inside packed column 14 is affected by pressure and temperature inside the column, length and diameter of the column, flowrate of the system, mobile phase composition, and composition and volume of the stationary phase inside the column. Collection system 16 can retain components of interest that correspond to detected fractionated peaks in the flowstream after column 14. Components that elute out of the column 14 can be detected by a detector 18 that receives a flowstream 20 split off from the primary flowstream 22. Collection system 16 collects purified fractions using a valve system 24 that is timed by a computer controller 26 to direct the flowstream 22 into collection system 16 when the flow corresponding to a peak reaches the valve 24. Flow not directed to collection system 16 passes to waste stream 28 or additional system processes. Controller 26 receives data from, and sends control signals to, pump 10, detector 18, and valve 24.
From the time a sample is injected into the column 14 at injector 12, each eluted component that is desirable for collection passes from the column 14 into the collection system 16 according to a timing calculation by controller 26 from the point of detection. This calculation accounts for the time for the detector 18 to analyze and process the flowstream, and the time for the peaks of eluted components to reach collection system 16. These timing factors are tracked by controller 26 that turns the valve 24 at the proper time to direct a fraction into the collection system 16 for the approximate length of time of the peak in the flowstream.
The detector 18 can be of two types, destructive or non-destructive. Ultraviolet detectors are commonly used non-destructive types and mass spectrometers are commonly used destructive types of detectors. Both types must be able to accurately detect the eluted compounds in the flowstream faster than the compounds will reach the collection system, otherwise some type of delay in the flowstream must be added. The detector can analyze and use sensors to detect the eluant compounds in the flowstream and send the signals to controller 26, which then triggers the valve 24 to time the direction of the flowstream into collection system 16 with the detected components. Detector 18 is upstream of the collection system and can be either in-line with the flowstream or split off 20 from the main flowstream line. A “splitter” divides a percentage of the entire mobile phase flow to the detector 18, while the remaining flow continues towards the collection system 16. A destructive tester 18 is usually installed on a split in the mobile phase flowstream.
U.S. Pat. No. 6,406,633 to Fischer describes timing of a fraction collection system after detection of sample fraction peaks. The timing calculation is determined empirically by injecting a calibrant into the mobile phase and detecting the calibrant using an upstream mass spectrometer split from the flowstream and a second downstream detector. The time of detection in the first detector and the time of detection of the same components in the second detector in the waste stream gives and actual time delay between those two points in the flowstream. In U.S. Pat. No. 6,997,031 to Gilly describes using configuration on an LC system to calibrate the system for delay and accurate fraction collection. The patent describes a mass spectrometer split off of the main flowstream prior to the collection system and a UV detector downstream of the collection system. Neither reference discloses, or even addresses, the problems associated with a delay caused by sensing and processing of an upstream detector and the travel time for a flowstream on a split to the detector prior to a collection system.
Although HPLC is an advancement over LC, similar problems exist for collection of sample fractions. Further, even more drawbacks exist to the current use of preparative HPLC. Elution periods ranging from several minutes to hours are necessary for each sample. Even in optimal conditions only a small fraction of the mobile phase contains components of interest. This can lead to very large volumes of waste mobile phase being generated in normal operation of the system.
For many applications, an alternative separation technology called supercritical fluid chromatography (SFC) has advanced past other chromatography technologies. SFC uses highly compressible mobile phases, which typically employ carbon dioxide (CO2) as a principle component. In addition to CO2, the mobile phase frequently contains an organic solvent modifier, which adjusts the polarity of the mobile phase for optimum chromatographic performance. Since different components of a sample may require different levels of organic modifier to elute rapidly, a common technique is to continuously vary the mobile phase composition by linearly increasing the organic modifier content. This technique is called gradient elution.
SFC has been proven to have superior speed and resolving power compared to traditional HPLC for many applications. This results from the dramatically improved diffusion rates of solutes in SFC mobile phases compared to HPLC mobile phases. Separations have been accomplished as much as an order of magnitude faster using SFC instruments compared to HPLC instruments using the same chromatographic column. A key factor to optimizing SFC separations is the ability to independently control flow, density and composition of the mobile phase over the course of the separation. SFC is finding significant advantages in the separation of enantiomers and is supplanting normal-phase HPLC for performing chiral separations.
A problem in all of the above-referenced separation technologies, but especially HPLC and SFC is when the chromatographic system is combined with a mass spectrometer on a split after the column but upstream from a collection system. The signal that is generated by the mass spectrometer, that indicates whether there is peak of a fraction ready to be collected, is generally delayed in time. The delay can last from a few hundred milliseconds up to ten seconds or more. Causes for the delay include operations for sensing, analyzing, and processing time in the mass spectrometer as well as the computer processing for a controller. These processing delays can be caused by complexity of the compound that creates the signal for the spectrometer to analyze, the complexity of rules for analyzing and presenting the signal, complexity or interference of other signals in the system caused by background interference from other components in the flowstream, and speed of the processor and bus in the instruments and in the controller. Other delays include transmission of the flowstream from the primary flow line 22 to the split detector 18 in flow line 20. In general, a signal from a detector may not vary widely for a particular sample run. However this is not always the case, and furthermore when beginning a sample run the delay is an unknown parameter that can adversely affect collection of the components. Changes in delay can be caused by numerous factors in the system that include flowrate and pressure, sample and mobile phase composition, and column 14 efficiency.
In LC and HPLC, a traditional system modification to compensate for this delay is to install a loop of tubing that is termed a “delay loop” in the industry. The delay loop 30, illustrated in FIG. 1, is placed after the split 20 to the detector 18 and prior to the flowstream reaching collection system 16. The delay loop 30 passes the primary flowstream through its coiled tubing. The length of the tubing adds a known amount of time after the detector has detected the component peak for the peak fraction to reach a valve decision point 24 that directs the flow to the collection device 16, a waste stream 28, or a different downstream process. Since a chromatography system flow typically includes an incompressible fluid, the delay can be calculated by volume divided by the flowrate.
A special problem in SFC in compensating for delay is that linear flowrates through tubes are generally significantly higher than HPLC and LC due to the extremely high flowstream pressures in SFC. The same problems apply to SFE. The use of wide-bore tubing in SFC is undesirable because of the problems associated with dispersion of the peak that would occur after the detector has defined the peak in the stream. Using small-bore tubing in SFC creates problems as well, which includes adding up to 20 or 30 meters of tubing for a delay compensation since the flowstream velocity is measured on the order of meters/second. Further, the extremely fast flowstream for a great distance can itself cause pressure and dispersion problems of the separated fraction in the flowstream. Further, on the low pressure side downstream of a BPR 37, the SFC flowstream undergoes up to a 500:1 expansion of the compressed gas in the mobile phase that is nonlinear throughout the tubing. The flowrate for this part of the flowstream is virtually impossible to calculate as the stream changes its composition within the tube.